Persistence of Transgene Expression in Synovium

滑膜中转基因表达的持久性

• 批准号: 6800409
• 负责人: Steven C Ghivizzani
• 金额: $ 29.27万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6800409
• 关键词: Adenoviridae; Lentivirus; arthritis; biotechnology; cytokine receptors; fibroblasts; flow cytometry; gene delivery system; gene expression; gene therapy; genetically modified animals; green fluorescent proteins; histocompatibility gene; immunogenetics; knee; laboratory rat; recombinant virus; synovial membrane; transfection; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Progress toward the clinical application of gene therapy for arthritis has been slowed by the inability to sustain intraarticular transgene expression. The prevailing observation is that transgene expression persists for only two to three weeks. The current proposal is designed to test the following hypotheses: 1) The immunologic incompatibility of foreign (non-self) transgene products leads to this premature termination of transgene expression, 2) Specific cell types within the synovium are capable of supporting stable, persistent transgene expression, and 3) In normal, immunocompetent animals, expression of cDNAs encoding homologous (self) or immunologically compatible gene products will persist long-term within the synovium. To determine the relative benefits and limitations of various ex vivo and in vivo gene delivery systems, each will be evaluated for transgenic persistence in the knees of athymic, nude rats. Because transduced cells that express foreign proteins survive for their natural life-span in this animal, it will serve as a "pseudohomologous" system within which we can use diagnostic marker genes to simulate the delivery of transgenes encoding "self" proteins to normal joints. Using the cDNA for Green Fluorescent Protein to phenotypically tag genetically modified cells in the synovium, we will use flow cytometry to characterize the populations of transduced cells. By studying how they change with time, we will identify the specific cell populations within the synovial lining that may permit long-term expression. The true motivation behind the study is however to determine for how long exogenous transgenes may be expressed in normal and arthritic joints in immunocompetent animals. For this, two different gene systems will be studied in normal rats that will not activate immune clearance. The first will be a soluble form of the rat TNF receptor type II; the second will be a "gene" construct that is transcribed, but not translated, avoiding altogether the immune issues of foreign protein products. The following specific aims will be addressed: 1) To comparatively evaluate synovial fibroblasts, dermal fibroblasts and mesenchymal stem cells for their capacity to enable persistent transgenic expression, and their biodistribution when used as vehicles for ex vivo intra-articular gene delivery to the knees of athymic nude rats, 2) To comparatively evaluate recombinant lentivirus, AAV and high capacity adenovirus for their capacity to enable persistent transgenic expression and their biodistribution when used as vehicles for direct intraarticular gene delivery to the knees of athymic nude rats, 3) To characterize genetically modified cell populations in the synovium and to determine how they change with time in vivo, and 4) Within immunocompetent rats, evaluate persistence of expression using immunocompatible transgene systems in normal and arthritic knees following ex vivo and in vivo delivery.

描述（由申请人提供）： 由于无法维持关节内转基因表达，基因治疗在关节炎中临床应用的进展已减慢。流行的观察结果是，转基因表达仅持续两到三周。 The current proposal is designed to test the following hypotheses: 1) The immunologic incompatibility of foreign (non-self) transgene products leads to this premature termination of transgene expression, 2) Specific cell types within the synovium are capable of supporting stable, persistent transgene expression, and 3) In normal, immunocompetent animals, expression of cDNAs encoding homologous (self) or immunologically compatible gene products will长期在滑膜内持续。为了确定各种离体和体内基因递送系统的相对益处和局限性，将评估每种裸体大鼠膝盖的转基因持久性。由于转导表达异物蛋白的转导细胞可为其在这种动物中的天然寿命中生存，因此它将用作“伪型”系统，在该系统中，我们可以使用诊断标记基因模拟编码“自我”蛋白质蛋白到正常关节的转基因的递送。使用绿色荧光蛋白的cDNA表型在滑膜中标记遗传修饰的细胞，我们将使用流式细胞仪来表征转导细胞的种群。通过研究它们如何随着时间的变化，我们将确定滑膜内部内部内部的特定细胞群体，这些细胞群可能会允许长期表达。但是，研究背后的真正动机是确定在免疫能力动物中可能在正常和关节炎关节中表达的外源性转基因的时间。为此，将在正常大鼠中研究两个不同的基因系统，这些基因系统不会激活免疫清除率。第一个将是大鼠TNF受体II型的可溶形式。第二个将是一种“基因”构建体，被转录但不翻译，从而完全避免了异物蛋白质产品的免疫问题。将解决以下具体目的：1）相对评估造纤维成纤维细胞，皮肤成纤维细胞和间质干细胞，以使其能够持续的转基因表达，并将其生物分配用作体内的体内基因时，将其用作自由度的锻炼，并评估较高的较高的较高的浓度和较高的浓度，以相比的浓度较高的浓度较高的浓度，可与较高的浓度相比，2）当用作直接直接的腹腔内基因递送到胸膜裸鼠的膝盖的车辆时，其能力的容量腺病毒能力能够启用持续的转基因表达及其生物分布在体内和体内递送后的关节炎膝盖和关节炎。